Autofocus algorithm for microscopy system based on cross-correlation

Abstract

Optimized automatic focusing of a microscope objective based on a cross correlation between a representative focus metric scan and a focus metric scan of a sample to be imaged.

The invention claimed is: 
     
       1. A method of an automated microscopy system automatically focusing a microscope objective, the method comprising:
 determining a focus position between the microscope objective and a reference microwell at a reference position on a microplate; 
 generating, by the automated microscopy system at a first time, an original focus metric scan of the reference microwell based on the focus position, the original focus metric scan comprising a first plurality of focus measure values of first images of the reference microwell respectively captured at a plurality of offsets in a direction of focus from the focus position, and each of the first plurality of focus measure values indicating a measurement of focus of a respective image among the first images; 
 storing the original focus metric scan in memory; 
 generating, by the automated microscopy system at a second time occurring after the first time, a current focus metric scan of a target microwell to be imaged on the microplate based on the focus position, the current focus metric scan comprising a second plurality of focus measure values of second images of the target microwell respectively captured at the plurality of offsets in the direction of focus from the focus position, and each of the second plurality of focus measure values indicating a measurement of focus of a respective image among the second images; 
 calculating, by the automated microscopy system, a cross correlation between the original focus metric scan and the current focus metric scan to determine an offset between the original focus metric scan and the current focus metric scan, wherein the focus position is moved to a position of the offset when the calculated cross correlation is low; and 
 determining, by the automated microscopy system, a corrected focus position along the focus axis to image a sample in the target microwell to be imaged using the focus of the microscope objective that is offset from the focus position based on the offset between the original focus metric scan and the current focus metric scan. 
 
     
     
       2. The method of  claim 1 , wherein the original focus metric scan is represented by a first two-dimensional curve that indicates a first relationship between (i) the first plurality of focus measure values and (ii) the plurality of offsets, and
 wherein the current focus metric scan is represented by a second two-dimensional curve that indicates a second relationship between (i) the second plurality of focus measure values and (ii) the plurality of offsets. 
 
     
     
       3. The method of  claim 2 , wherein the cross correlation comprises a correlation between the first two-dimensional curve and the second two-dimensional curve. 
     
     
       4. The method of  claim 3 , wherein the correlation is an alignment of the first plurality of focus measure values captured at the offsets in the direction of focus to the second plurality of focus measure values captured at the offsets in the direction of focus. 
     
     
       5. The method of  claim 4 , wherein the determining the focus position comprises one of a user manually determining the focus position and the automated microscopy system automatically determining the focus position by performing a scan of images and selecting based on the scan of images a focus position of a best focused image as the focus position. 
     
     
       6. A method of an automated microscopy system automatically focusing a microscope objective, the method comprising:
 determining a focus position between the microscope objective and a reference microwell at a reference position on a microplate; 
 generating, by the automated microscopy system at a first time, an original focus metric scan of the reference microwell based on the focus position, the original focus metric scan comprising a first plurality of focus measure values of first images of the reference microwell respectively captured at a plurality of offsets in a direction of focus from the focus position, and each of the first plurality of focus measure values indicating a measurement of focus of a respective image among the first images; 
 storing the original focus metric scan in memory; 
 generating, by the automated microscopy system at a second time occurring after the first time, a current focus metric scan of a target microwell to be imaged on the microplate, the current focus metric scan comprising a second plurality of focus measure values of second images of the target microwell respectively captured at the plurality of offsets in the direction of focus from the focus position, and each of the second plurality of focus measure values indicating a measurement of focus of a respective image among the second images; 
 calculating, by the automated microscopy system, a cross correlation between the original focus metric scan and the current focus metric scan to determine an offset between the original focus metric scan and the current focus metric scan, wherein the focus position is moved to a position of the offset when the calculated cross correlation is low; and 
 determining, by the automated microscopy system, a corrected focus position along the focus axis to image a sample in the target microwell to be imaged using the focus of the microscope objective that is offset from the focus position based on the offset between the original focus metric scan and the current focus metric scan. 
 
     
     
       7. The method of  claim 6 , wherein the original focus metric scan is represented by a first two-dimensional curve that indicates a first relationship between (i) the first plurality of focus measure values and (ii) the plurality of offsets, and
 wherein the current focus metric scan is represented by a second two-dimensional curve that indicates a second relationship between (i) the second plurality of focus measure values and (ii) the plurality of offsets. 
 
     
     
       8. The method of  claim 7 , wherein the cross correlation comprises a correlation between the first two-dimensional curve and the second two-dimensional curve. 
     
     
       9. The method of  claim 8 , wherein the correlation is an alignment of the first plurality of focus measure values captured at the offsets in the direction of focus to the second plurality of focus measure values captured at the offsets in the direction of focus. 
     
     
       10. The method of  claim 9 , wherein the determining the focus position comprises one of a user manually determining the focus position and the automated microscopy system automatically determining the focus position by performing a scan of images and selecting based on the scan of images a focus position of a best focused image as the focus position.